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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李佳音 | |
| dc.contributor.author | San-Chi Chang | en |
| dc.contributor.author | 張珊齊 | zh_TW |
| dc.date.accessioned | 2021-06-16T10:13:28Z | - |
| dc.date.available | 2023-08-19 | |
| dc.date.copyright | 2013-08-28 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60191 | - |
| dc.description.abstract | 腸炎弧菌(Vibrio parahaemolyticus)又稱為副溶血弧菌，是一株嗜鹽性的革蘭氏陰性菌，為人類的病原菌。其胞外鹼性絲胺酸蛋白酶，PrtA (VPA0227)為腸炎弧菌可能的致病因子之一。鹼性絲胺酸蛋白酶於菌體生長至後對數期時大量分泌，PrtA的表現受到細胞間訊息傳遞之定額感應系統 (quorum sensing system) 的正調控，主要的定額感應調控子為OpaR。本研究預測prtA的上游區域有兩個可能為OpaR的結合位置–3~–145及–229~–361，經由電泳遷移實驗(EMSA)的實驗結果確認OpaR可以結合於此二區域。在EMSA實驗過程中添加廣泛調控菌株的第二傳訊者c-di-GMP，可以觀察到c-di-GMP阻擾OpaR和prtA的結合。此外，本研究探討主要掌管菌株生長穩定期的sigma factor RpoS對於PrtA分泌的影響。本研究以同源重組的方式建構突變株ΔopaR、 ΔrpoS 和ΔopaRΔrpoS，發現NO.93和ΔopaR的生長速率並無明顯差異，然而ΔrpoS 和ΔopaRΔrpoS的生長速率則較NO.93低。透過即時定量聚合酶連鎖反應法(QPCR)觀察野生株NO.93中各目標基因的表現情形，RpoS於菌株培養至穩定生長期時達最高表現量; 定額感應的主要調控子OpaR則於菌株存在高細胞密度(OD600 0.1 )後穩定表現。然而，磷脂質依存溶血素LDH及PrtA則分別於初對數期和後對數期時大量表現。由QPCR及西方墨點法證實RpoS和OpaR 對於prtA基因表現量以及胞外PrtA蛋白表現量為正調控，並且影響胞外PrtA的分泌時間。此發現有助於未來其他具有細胞毒性的蛋白酶探討其致病機制與調控方式。 | zh_TW |
| dc.description.abstract | Vibrio paraheamolyticus is a human pathogen, it cause gastroenteritis and diarrhea in Taiwan, Asia and coast area. It is a gram-negative bacterium. Its extracellular alkine serine protease, PrtA (VPA0227) was seen to be a virulence factor of Vibrio paraheamolyticus No. 93. The extracellular alkine serine protease, PrtA get to maximal expression when growing to late log phase. We know the expression of PrtA would upregulated by quorum sensing system, and the major quorum sensing in Vibrio paraheamolyticus is OpaR. In this study, there are two probable OpaR binding sites upstream of prtA, one located on –3~–145 and another on –229~–361. We purify the OpaR protein and do EMSA to confirm that OpaR would regulate prtA by binding on its promoter region directly. And the second messenger molecule c-di-GMP would interfere with the binding between the OpaR and prtA. Besides, we use homologous recombination to make the deletion mutants ΔopaR, ΔrpoS, ΔopaRΔrpoS for the further studies. We found the growth rate of VP93 and ΔopaR are the same, however ΔrpoS and ΔopaRΔrpoS are lower than VP93. The results from Q-PCR show that the prtA was expressed to the most high level when VP93 was cultured at 3 hour; rpoS was 8 hour ; the ldh was at 1.5 hour. And the quorum sensing regulator opaR express stablely from 1.5 to 12 hour. From the result of Q-PCR and Western, the OpaR and RpoS can promote the expression and secretion of extracellular serine protease PrtA of V. parahaemolyticus NO.93. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T10:13:28Z (GMT). No. of bitstreams: 1 ntu-102-R00623008-1.pdf: 3576328 bytes, checksum: 7baf49cd5b5eb995307e1ccb6a1a6881 (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 中文摘要 iv 英文摘要 v 目錄 vi 表次 x 圖次 xi 附錄圖次 xiii 縮寫表 xiv 壹、前言 1 一、腸炎弧菌(Vibrio parahaemolyticus) 1 1. 概述 1 2. 性狀 2 3. 基因體分析 2 4. 毒力分析 3 二、胞外絲胺酸蛋白酶 4 1. 概述 4 2. 腸炎弧菌(Vibrio parahaemolyticus)胞外絲胺酸蛋白酶 5 3. 霍亂弧菌(Vibrio cholera)胞外絲胺酸蛋白酶 5 4. 創傷弧菌(Vibrio vulnificus)胞外絲胺酸蛋白酶 6 三、定額感應系統(Quorum Sensing System) 8 1. 概述 8 2. 哈維氏弧菌(Vibrio harveyi)定額感應系統概述 8 3. 哈維式弧菌(Vibrio harveyi) LuxR之研究 9 4. 腸炎弧菌(Vibrio parahaemolyticus) OpaR之研究 10 5. 霍亂弧菌(Vibrio cholera) HapR之研究 11 6. 創傷弧菌(Vibrio vulnificus) SmcR之研究 11 四、腸炎弧菌c-di-GMP的相關研究 12 1. 概述 12 2. c-di-GMP調控鞭毛的相關研究 12 3. c-di-GMP調控莢膜多醣的相關研究 13 五、廣泛壓力調控因子RpoS 14 1.概述 14 2. RpoS調控創傷弧菌(Vibrio vulnificus)的毒力因子 16 3. RpoS調控霍亂弧菌(Vibrio cholera)的毒力因子 16 3. RpoS調控腸炎弧菌(Vibrio paraheamolyticus)的毒力因子 18 六、研究目的與動機 19 貳、 實驗材料與方法 20 I. 實驗材料 20 一、實驗菌株、質體和引子 20 二、培養基及抗生素使用量 20 三、化學藥品及試劑 20 四、儀器設備 21 五、各種實驗溶液 22 II. 實驗方法 25 一、DNA技術 25 二、RNA技術 30 三、蛋白質技術 32 四、建構ΔopaR及ΔopaRΔrpoS突變株 35 五、細菌生長曲線之測定 37 六、胞外蛋白酶活性之測定 38 七、電泳遷移實驗EMSA (Electrophoretic mobility shift assay) 38 八、統計分析方法 41 参、 實驗結果 42 一、PrtA胺基酸序列分析 42 二、prtA上游核酸序列之OpaR結合區位的預測 42 三、以EMSA證實OpaR蛋白和PrtA上游片段直接結合 43 四、c-di-GMP會影響OpaR和PrtA上游核酸的結合 44 五、建構腸炎弧菌NO.93之ΔopaR突變株和ΔopaR rpoS雙突變株 45 六、菌株生長曲線 46 七、以Q-PCR觀察opaR、rpoS、 ldh以及prtA隨時間的轉錄情形 46 八、以Q-PCR比較各菌株間prtA以及ldh基因轉錄之差異 47 九、觀察腸炎弧菌各菌株的胞內外蛋白酶PrtA隨時間的分泌情形 48 1. Wild type (VP93) 胞內外PrtA和時間關係 48 2. ΔopaR胞內外PrtA和時間關係 49 3. ΔrpoS胞內外PrtA和時間關係 49 4. ΔopaRΔrpoS胞內外PrtA和時間關係 49 十、比較腸炎弧菌NO. 93以及各突變菌株的胞內外蛋白酶PrtA的分泌差異 50 十一、比較腸炎弧菌NO. 93各基因型菌株胞外蛋白酶活性 50 肆、 討論 52 (1) 定額感應的主要調控子OpaR對於腸炎弧菌PrtA以及LDH表現及分泌路徑的調控 52 (2) 第二傳訊者c-di-GMP對於腸炎弧菌PrtA表現及分泌路徑的調控 52 (3)穩定生長期主要表現的Sigma factor RpoS對於蛋白酶PrtA分泌量之相關趨勢 53 伍、 結論 54 陸、 文獻參考 56 | |
| dc.language.iso | zh-TW | |
| dc.subject | 腸炎弧菌 | zh_TW |
| dc.subject | 絲胺酸蛋白酶 | zh_TW |
| dc.subject | Vibrio parahaemolyticus | en |
| dc.subject | serine protease | en |
| dc.subject | virulence | en |
| dc.title | "腸炎弧菌絲胺酸蛋白酶PrtA受C-di-GMP, OpaR和RpoS調控之研究" | zh_TW |
| dc.title | The regulation of Vibrio parahaemolyticus serine protease PrtA by cyclic diguanylate, OpaR, and RpoS | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 劉瑞芬,林榮流 | |
| dc.subject.keyword | 腸炎弧菌,絲胺酸蛋白酶, | zh_TW |
| dc.subject.keyword | Vibrio parahaemolyticus,serine protease,virulence, | en |
| dc.relation.page | 105 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-08-20 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| 顯示於系所單位： | 農業化學系 | |
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